Project Summary Stevens Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) is a severe drug hypersensitivity reaction manifesting as sloughing of skin, eyes, mouth, throat and genitals, along with systemic inflammation and internal organ involvement. Disease can occur in otherwise healthy people and is largely unpredictable. Mortality is 20- 30% and there is no known treatment. SJS/TEN is a substantial burden on the US healthcare system, costing an estimated 129 million dollars per year. It is of paramount importance to elucidate the pathobiology of SJS/TEN to improve disease prevention, diagnosis and treatment. Despite this clear need, SJS/TEN is significantly under-researched. The dearth of high-quality research in this field stems from two major barriers: a lack of patient samples available for analysis and the absence of an easily attainable and reproducible model system to perform mechanistic studies. One major gap in our knowledge of the pathogenesis of SJS/TEN is the putative mechanism(s) by which immune cells mediate keratinocyte death. It is largely assumed that cytolytic granules generated by cytotoxic CD8+ T cells induce keratinocyte apoptosis. However, contradictory findings call this assumption into question. The central hypothesis of this proposal is that the inflammatory cell death pathways necroptosis and pyroptosis, rather than apoptosis, mediate keratinocyte death and epidermal destruction in SJS/TEN. The proposed study will directly test this hypothesis while overcoming current barriers in the field. Aim 1 employs two novel technologies to study a large bank of formalin-fixed paraffin embedded clinical specimens that until now were of limited research utility. Clinical specimens are drawn from a meticulously developed and thoroughly vetted patient database that spans three major tertiary care hospitals. These technologies are (i) OpalTM multispectral immunofluorescence staining with MantraTM Quantitative Pathology Imaging System and inFORM Image Analysis Software and (ii) NanoString NCounter platform for gene expression profiling. Aim 2 interrogates mechanism(s) of keratinocyte death by partnering with burn teams at two major centers to prospectively collect blister fluid and affected epidermis, a unique sample resource. Analysis utilizes RNAseq, high parameter flow cytometry, and multiplex immunoassay in concert with traditional laboratory techniques. Aim 3 generates a novel ex vivo model of SJS/TEN using patient blister fluid with commercially available 3D full thickness tissue engineered human skin that will be used to investigate the proposed hypothesis while also exploring potential novel treatments. The data generated through this innovative research has significant potential to advance understanding of fundamental disease pathobiology and clinical care.